The present invention relates to a copper tolerant yeast, the use of the yeast to recover copper from a solution and a pectinase produce by the yeast and the use of the pectinase.
The present invention also relates to a method of hydrolyzing pectins contained in the pectin-containing substrates. The present invention also relates to a method of producing galacturonate.
The influences of mine-pollution have been argued since the damages caused by the mine-pollution were spread out because of the wastewater flew into rivers. The damages include Ashio copper mine pollution injury (about 1980), Minamata disease caused by methyl mercury (1950's) and Itai-Itai disease (about 1940). However, it is not well known how the heavy metals affect organisms at the cellular levels. Those firstly affected by heavy metals under the natural environment are microorganisms. Many heavy metal tolerant strains are obtained to reveal the influence of heavy metals on organisms and to depollute the polluted area of rivers or lands. Many of these strains were bacterial strains.
Among the heavy metal tolerance, many reports have been published about copper tolerant microorganisms and their copper tolerance. For Escherichia coli, some plasmids are involved in copper tolerance by reducing Cu(+2) to Cu(+1) (Brown et al., 1992). A copper tolerant strain has also been obtained for budding yeast S. cerevisiae, wherein the strain acquired the copper tolerance to about 1 mM copper by detoxicating Cu(+2) to insoluble CuS by producing large amount of H2S (Ashida, 1965). Thus, the mechanisms known in the prior art are based on the reduction of Cu(+2) to Cu(+1) after incorporating copper ion into cells. Many of these investigations are carried out on bacteria, and indeed, many microorganisms which have been utilized are bacteria. The foregoing report for S. cerevisiae is one of the limited examples of these investigations on eukaryotes.
Yeast is the representative example of eukaryotes, and is an important microorganism for investigating the influences of heavy metals on human beings which is another representative example of eukaryotes. The sources for isolating yeasts are widely spread, including the surfaces or internals of animals or plants, soils, atmosphere and rivers. Although there are few examples where yeasts were isolated from deep-sea, yeasts were isolated from deep-sea sediments collected by manned submersibles “SHINKAI 2000” and “SHINKAI 6500”. To date, 24 of deep-sea yeast strains were also isolated from deep-see sediments of Sagami Bay (1100-1400 m depth) and 13 strains were isolated from Japan Trench(4500-6500 m depth). The features of yeasts living in such a special environment have not been sufficiently revealed, because it has been difficult to obtain such yeasts themselves. Furthermore, it has been unknown about what kind of enzymes such yeast may secrete.
Generally, many applications of enzymes are known in the fields of foods or cosmetics. Especially, in the field of food industry, pectinases, polygalacturonases and pectin liases, which hydrolyze pectins, have been used for producing clear fruit juice or as a tool for removing endocarps of citruses. Pectin is an acidic polysaccharide which is unique to the non-lignified tissues and is known to be present in the peel of citrus fruits or fruits such as apple and constitutes the cell walls or intercellular substances in the form associated with galactan or arabinan. In the field of food industry where fruits are use as materials, a large amount of peels removed to obtain pulps should be discarded, which raised some problems from the viewpoint of cost for processing them and the effective utilization of resources. Furthermore, the effective utilization of resources is also desired because galacturonate, which is the hydrolyzed product of pectin, may be used during food processing as an acidifier and it is notable as diet foods because of their low calorie. It can be also noted that the effective utilization of resources has been insufficient in that vitamins are discarded with peels, although peels contain larger amount of vitamins than pulps in general. Therefore, a method for efficiently decomposing pectins has been desired. Especially, pectinases having high activity under wide range of conditions and methods for providing such pectinases in large scale have been strongly desired.
There have been several reports on yeast pectinases. For examples, the pectinase secreted by budding yeast Saccharomyces cerevisiae is known to have the optimum pH of 5.5 and the optimum temperature of 45° C. (Blanco, et al., 1994). On the other hand, there are fewer reports for pectinases secreted by Cryptococcus albidus. Brown et al. (1985) describes that the pectinase secreted by Cry. albidus had a molecular weight of 41,000 and had the optimum pH of 3.7 and optimum temperature of 37° C. Previously known pectinases loose almost all the activities in the presence of Hg2+, Cu2+, Fe2+, and Al3+ due to the denaturation of proteins
However, there have been no reports on whether the pectin hydrolyzing enzymes are produced and secreted by such deep-sea yeasts and there have been also no reports on the physiochemical and biochemical characteristics of such enzymes.